Methods of screening compounds for GRK6 modulating activity

Abstract

The present invention relates to methods of treating disease by altering G protein coupled receptor kinase (GRK) 6. This may be done by altering the expression or activity of the protein, for example. The present invention may be used for disease diagnosis, by detecting the expression or activity of GRK6. The present invention relates to a GRK6 deficient mouse, GRK6 splice variants, and methods of use. The present invention also relates to methods of identifying compounds that alter GRK6 activity. The present invention relates to disease treatment by altering GRK6 expression or activity.

1 . A method of screening for modulators of GRK6-associated desensitization comprising: 
 (a) providing a cell comprising a GRK6 and a GPCR;    (b) contacting said cell with a candidate modulator; and    (c) monitoring said cell for GRK6-associated desensitization.    
     
     
         2 . The method of  claim 1 , wherein the monitoring comprises determining the cellular distribution of the GRK6, GPCR, or arrestin.  
     
     
         3 . The method of  claim 1 , wherein the GRK6 is a modified GRK.  
     
     
         4 . The method of  claim 3 , wherein the GRK6 includes a CAAX motif.  
     
     
         5 . A method for identifying compounds that modulate GRK6 comprising the steps of: 
 (a) providing a cell comprising GRK6, a GPCR, and an arrestin, and wherein at least one of said molecules is detectably labeled;    (b) exposing the cell to the compound(s);    (c) determining the cellular distribution of the GRK6, GPCR, or arrestin;    (d) comparing the cellular distribution of the GRK6, GPCR, or arrestin in the presence of the compound(s) to the cellular distribution of the GRK6, GPCR, or arrestin in the absence of the compound(s); and    (e) correlating a difference between (1) the cellular distribution of the GRK6, GPCR, or arrestin in the presence of the compound(s) to (2) the cellular distribution of the GRK6, GPCR, or arrestin in the absence of the compound(s) to modulation of GRK6 activity.    
     
     
         6 . The method of  claim 5 , wherein the GRK6 is overexpressed.  
     
     
         7 . The method of  claim 5 , wherein the labeled molecule is localized in the cytosol, plasma membrane, clathrin-coated pits, endocytic vesicles or endosomes.  
     
     
         8 . The method of  claim 5 , wherein the detectable molecule is a radioisotope, an epitope tag, an affinity label, an enzyme, a fluorescent group, or achemiluminescent group.  
     
     
         9 . The method of  claim 5 , wherein the molecule is detectably labeled due to its interaction with another molecule, which may be detectably labeled.  
     
     
         10 . The method of  claim 5 , wherein the GRK6 is a modified GRK.  
     
     
         11 . The method of  claim 10 , wherein the GRK6 includes a CAAX motif.  
     
     
         12 . A method for inhibiting desensitization of the dopamine receptor in cell comprising contacting the cell with a compound that decreases GRK6 activity or the expression of a nucleic acid encoding GRK6.  
     
     
         13 . The method of  claim 12 , wherein the compound is an antisense oligonucleotide.  
     
     
         14 . The method of  claim 13 , wherein the antisense oligonucleotide inhibits expression of a nucleic acid encoding GRK6.  
     
     
         15 . A method for treating diseases involving the dopamine receptor, wherein the effectiveness of endogenous dopamine is increased by altering GRK6 activity or expression.  
     
     
         16 . The method of  claim 15 , wherein the disease is Parkinson's, schizophrenia, Tourette Syndrome, depression, or drug-addiction.  
     
     
         17 . A method of modulating desensitization of a dopamine receptor in a cell, comprising: 
 (a) providing a cell expressing a dopamine receptor and a G protein coupled receptor kinase (GRK);    (b) modulating the activity of the GRK; and    (c) exposing said cell to an agonist.    
     
     
         18 . The method of  claim 17 , wherein the GRK is GRK6.  
     
     
         19 . The method of  claim 18 , wherein the expression of GRK6 is increased.  
     
     
         20 . The method of  claim 18 , wherein the expression of GRK6 is decreased.  
     
     
         21 . The method of  claim 18 , wherein the activity of GRK6 is increased.  
     
     
         22 . The method of  claim 18 , wherein the activity of GRK6 is decreased.  
     
     
         23 . A method of treating a disease by modulating desensitization of a dopamine receptor in a host cell, comprising: (a) providing a compound which modulates the expression or activity of a GRK6; and (b) administering said compound to a host.  
     
     
         24 . The method of  claim 23 , wherein said method comprises concurrent administration of the compound that modulates expression or activity of a GRK6 with a compound that modulates a G-protein coupled receptor.  
     
     
         25 . A nucleic acid having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.  
     
     
         26 . A nucleic acid having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.  
     
     
         27 . A vector comprising a nucleic acid having a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.  
     
     
         28 . The vector of  claim 27 , wherein the nucleic acid is flanked by IoxP sites.  
     
     
         29 . A host cell comprising a nucleic acid having the nucleic acid sequence of SEQ ID No: 19.  
     
     
         30 . An isolated immunoglobulin which recognizes and binds to a GRK, or fragment thereof.  
     
     
         31 . The immunoglobulin of  claim 30 , wherein the GRK6 is GRK6a, GRK6b, GRK6c, or GRK6d.  
     
     
         32 . The immunoglobulin of  claim 30 , wherein the GRK fragment has the sequence of SEQ ID No. 3.  
     
     
         33 . The immunoglobulin of  claim 30 , wherein the antibody fragment is Fab, Fab′, F(ab′)2, F(v),and scFv.  
     
     
         34 . A method of detecting GRK6 in a biological sample, comprising: (a) exposing the biological sample to an immunoglobulin of  claim 30;  and (b) determine whether the immunoglobulin bound a protein of the biological sample.  
     
     
         35 . The method of  claim 34 , wherein the binding of the immunoglobulin to the protein indicates the presence of or predisposition to a disease.